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Mesenchymal Stem Cell-Derived Microvesicles Improve Intestinal Zheng et al. Stem Cell Research & Therapy (2021) 12:299 https://doi.org/10.1186/s13287-021-02363-0 RESEARCH Open Access Mesenchymal stem cell-derived microvesicles improve intestinal barrier function by restoring mitochondrial dynamic balance in sepsis rats Danyang Zheng, Henan Zhou, Hongchen Wang, Yu Zhu, Yue Wu, Qinghui Li, Tao Li*† and Liangming Liu*† Abstract Background: Sepsis is a major cause of death in ICU, and intestinal barrier dysfunction is its important complication, while the treatment is limited. Recently, mesenchymal stem cell-derived microvesicles (MMVs) attract much attention as a strategy of cell-free treatment; whether MMVs are therapeutic in sepsis induced-intestinal barrier dysfunction is obscure. Methods: In this study, cecal ligation and puncture-induced sepsis rats and lipopolysaccharide-stimulated intestinal epithelial cells to investigate the effect of MMVs on intestinal barrier dysfunction. MMVs were harvested from mesenchymal stem cells and were injected into sepsis rats, and the intestinal barrier function was measured. Afterward, MMVs were incubated with intestinal epithelial cells, and the effect of MMVs on mitochondrial dynamic balance was measured. Then the expression of mfn1, mfn2, OPA1, and PGC-1α in MMVs were measured by western blot. By upregulation and downregulation of mfn2 and PGC-1α, the role of MMVs in mitochondrial dynamic balance was investigated. Finally, the role of MMV-carried mitochondria in mitochondrial dynamic balance was investigated. Results: MMVs restored the intestinal barrier function by improving mitochondrial dynamic balance and metabolism of mitochondria. Further study revealed that MMVs delivered mfn2 and PGC-1α to intestinal epithelial cells, and promoted mitochondrial fusion and biogenesis, thereby improving mitochondrial dynamic balance. Furthermore, MMVs delivered functional mitochondria to intestinal epithelial cells and enhanced energy metabolism directly. Conclusion: MMVs can deliver mfn2, PGC-1α, and functional mitochondria to intestinal epithelial cells, synergistically improve mitochondrial dynamic balance of target cells after sepsis, and restore the mitochondrial function and intestinal barrier function. The study illustrated that MMVs might be a promising strategy for the treatment of sepsis. Keywords: Mesenchymal stem cell-derived microvesicles, Sepsis, Intestinal barrier function, mfn2, PGC-1α, Mitochondrial dynamic balance * Correspondence: [email protected]; [email protected] †Tao Li and Liangming Liu contributed equally to this work. State Key Laboratory of Trauma, Burns and Combined Injury, Shock and Transfusion Department, Research Institute of Surgery, Daping Hospital, Army Medical University, Daping, Chongqing 400042, People’s Republic of China © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zheng et al. Stem Cell Research & Therapy (2021) 12:299 Page 2 of 21 Introduction Under physiological circumstances, mitochondria are in Sepsis is a severe host response disorder to infection, a dynamic balance of continuous fission and fusion [22, which is an important cause of multiple organ dysfunc- 25], while the balance would be broken during sepsis, tion and a common clinical severe disease [1, 2]. Ac- which manifests as excessive mitochondrial fission and cording to epidemiological data in 2016, the total insufficient mitochondrial fusion, along with the break number of sepsis patients was 31.5 million in the globe, of cristae and the decrease of mitochondrial quality [22, and the number has rapidly risen to 48.9 million in 25, 26]. The imbalance of mitochondrial dynamics could 2020, which suggested that sepsis was still a huge burden lead to the generation of ROS and the release of for global health [1–3]. The intestinal barrier plays an mtDNA, which is a major reason for cellular dysfunction essential role in absorbing nutrients and preventing bac- [25–27]. Therefore, we further speculate that MMV teria and toxins from the intestinal lumen [4, 5], and the might restore the function of intestinal epithelial cells to intestine has been considered as the motor of multiple improve the intestinal barrier function by improving organ dysfunction syndromes during sepsis for a long mitochondrial dynamic balance after sepsis. To test this time, which allows the translocation of bacteria and hypothesis, MMVs were used to treat sepsis rats and in- toxins from the intestinal lumen to circulation after testinal epithelial cells (IEC-6), and the role and the damage [6], and in turn aggravates the progress of sep- underlying mechanism of MMVs on mitochondrial dy- sis, while the effective treatment is absent [7]. namic balance and intestinal barrier function were ob- Microvesicles (MVs) are natural membranous vesicles served in the present study. carrying a lot of biologically active molecules such as an- tigens, growth factors, mRNAs, and lipids, which are Materials and methods proved to participate in the regulation of various patho- Ethics approval and sepsis procedure physiological processes [8–10]. Stem cell-derived MVs The animal use and operation procedures were approved are emerging therapeutic strategies for many diseases by the Ethics Committee of the Army Medical Center due to the MV-carried bioactive substances and the low (Daping Hospital, Army Medical University, Chongqing, immunogenicity [9–12]. For example, MSC-derived China, No. DHEC-2012-069). Sprague-Dawley (SD) rats MVs (MMVs) were proved as an effective treatment in of either sex (weighting 190–215g) were provided by the severe bacterial pneumonia by delivering keratinocyte Animal Center of the Army Medical Center. The cecal growth factor to inflammatory cells [13]. MMVs were ligation and puncture (CLP) procedure was induced as able to promote the recovery of acute kidney injury by described previously [28, 29]. Briefly, rats were anesthe- improving the proliferation and inhibiting the apoptosis tized by sodium pentobarbital (25mg/kg, IP), and a 2-cm in tubular cells [14]. Studies also showed that stem cell- incision was made along the linea alba, then the end of derived MVs could be used in the treatment of gastro- the cecum was exposed and ligated, and the ligated intestinal diseases. MSC-derived extracellular vesicles cecum was punctured by a triangular pyramid, and the could polarize macrophages and attenuated the colonic above operations were carried out under aseptic condi- inflammatory response in inflammatory bowel disease tions. After the operation, rats were allowed water and and protected the function of the gut barrier by transfer- food ad libitum. ring metallothionein-2 [15]. Therefore, we hypothesized that MMVs might improve sepsis-induced intestinal bar- Cell culture and agents rier dysfunction, which might be related to the thera- MSCs were extracted from the tibia and femoral bone mar- peutic proteins carried in MMVs. row of weaned SD rats (weighting 45–75g, 3–4weeksold). Previous studies showed that MMVs usually exerted In detail, the tibia and femur were collected after rats were therapeutic effects via modulating genes and proteins in sacrificed under aseptic conditions. Then, the bones were target cells [13, 14, 16–19], and recent studies suggested washed 5 times with sterile PBS, and the bone marrow was that MMVs might also improve the function of organ- extracted by flushing the bones with 6mL MesenCultTM elles as mitochondria. For example, MMVs could attenu- Expansion Kit media (Stemcell Technologies, Canada), sup- ate mitochondrial damage to alleviate inflammation in plied with 80U/ml penicillin (Gibco, America) and 2mM L- acute kidney injury [20], and improve mitochondrial Glutamine (Stemcell Technologies, Canada) [30]. Then, the fragmentation and ATP generation to promote the re- mixed medium was cultured at 37°C and 5% CO2,andcells covery of kidney function [21]. Mitochondria are import- at 3–5 passages were used to harvest MMVs. IEC-6 was ant organelles in cells, which provide energy for various purchased from ATCC (America) and was cultured in physiological processes [22, 23]. In addition to energy DMEM high glucose medium with 10% fetal bovine serum metabolism, mitochondria are also involved in a variety (Gibco, America) and 80U/ml penicillin (Gibco, America). of pathophysiological processes, such as calcium ion Chondrocytes were obtained from the cartilage of the hip homeostasis, cell apoptosis, and proliferation [22–24]. and knee joints of SD rats (5–6 weeks old). In detail, the Zheng et al. Stem Cell Research & Therapy (2021) 12:299
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